Process for preparing polynucleotides on a solid support in a tightly packed bed

ABSTRACT

The present invention relates to a process for preparing polynucleotides on a solid support in a reactor in the form of a column through which solutions of reagents and/or solvents are circulated, wherein the solid phase constituting said solid support is immobilized in said reactor, and said solutions migrate in the column and through the solid phase according to a frontal progression, such that the successive solutions from each step of a synthesis cycle do not mix at all, or very little. 
     The subject of the present invention is also a reactor consisting of a column completely filled with particles of porous materials constituting the solid support, and a synthesis device including such a reactor.

The present invention relates to a process for preparing polynucleotideson a solid support. The present invention also relates to a reactorcontaining a solid support and to a device including this reactor, whichare useful in the process for preparing polynucleotides according to theinvention.

The synthesis of polynucleotides on a solid support is particularly usedin automated syntheses of DNA or RNA oligonucleotides. In the presentapplication, "polynucleotides" is understood to mean deoxyribonucleicacid or ribonucleic acid fragments or, more generally, polynucleotidesor oligonucleotides where the bases, inter-nucleotide phosphatelinkages, or alternatively the ribose rings of the bases, can bechemically modified in a known manner. This may be especiallyoligonucleotides with α or β anomers, oligonucleotides withinter-nucleotide linkage of the phosphorothioate or methyl phosphonatetype, or alternatively oligothionucleotides.

The aim of the invention is the improvement of the efficiencies ofexisting methods of synthesis of oligonucleotides.

The method according to the present invention consists in a modificationof organic synthesis apparatuses and parameters so as to improve theproductivity thereof.

The improvements affect in particular the duration and the consumptionof reagents necessary for carrying out a synthesis.

The principle for the chemical synthesis of nucleic acids on a solidsupport is nowadays widely described in the specialist literature, and anumber of apparatus are available on the market which performautomatically all or part of the synthesis steps. Among the chemicalroutes described, only the so-called phosphoramidites method (Carutherset col: EP 0,035,719 B1) is up until now sufficiently efficient toenvisage the production of nucleic acids on an industrial scale.

The preparation of oligonucleotides or polynucleotides is carried out ina reactor containing a solid support and comprises the treatment of thesolid support such as an inorganic polymeric support by a series ofsuccessive steps, each of the series leading to the addition of a newnucleotide on the support. The series of successive steps, or synthesiscycles, are carried out as many times as is required by the manufactureof an oligonucleotide or a polynucleotide of desired length.

In a process for synthesizing polynucleotides on a solid support, thesolid support traditionally consists of glass beads of controlledporosity (CPG) or, more generally, of particles of a functionalizedinorganic or organic polymer.

The techniques conventionally used involve the use of eight differentreagents as solid supports, consisting of the said functionalizedinorganic or organic polymer linked to a nucleoside A, T, C, G or U,depending on whether the sequence to be prepared contains, as firstdeoxyribo- or ribonucleotide A, T, C, G or U. Manufacturers thereforesupply reactors in which one of these nucleosides has previously beenattached to the support. Depending on whether the sequence starts withA, T, C, G or U, the appropriate reactor is then chosen. The elongationof this first nucleoside then occurs in the 3'→5', or 5'→3' direction,using coupling reagents.

Numerous supports have already been described in the literature for thesolid phase synthesis of oligonucleotides.

There may be mentioned organic polymers such as polystyrene (Nucleic A.Res. 1980, volume 8), polyacrylamide acryloylmorpholide,polydimethylacrylamide polymerized on kieselguhr (Nucleic Ac. Res. 9(7)1691 (1980)).

Other supports described are of inorganic nature, in particular based onsilica functionalized by a hydrocarbon radical carrying an NH₂ and/orCOOH group (J. Am. Chem., 105, 661 (1983)), or the support based onsilica functionalized by a 3-aminopropyltriethoxysilane group whose usein phosphite and phosphoramidite synthesis for the preparation ofoligonucleotides was described for the first time in European Patent No.0,035,719.

There is known, from French Patent Application FR 93 08 498 andPCT/FR94/00842, a process for the solid phase synthesis ofoligonucleotides in which a so-called "universal" support is used, thatis to say a solid support which can be used regardless of the firstnucleotide of the RNA or DNA to be synthesized, regardless of the typeof monomeric reagent used during the synthesis, that is to sayregardless of the type of substitution on the phosphate group in 3' orin 5', depending on whether the synthesis is carried out in the 5'→3' or3'→5' direction.

In particular, the "universal nature" of the solid phase supports can beobtained by functionalization of the inorganic or organic polymer with ahydrocarbon radical containing glycol type groups in which an OH groupand a nucleophilic group are present in the vicinal position, that is tosay on two adjacent carbons, at the end of the hydrocarbon radical, itbeing possible for these two carbons to be optionally substituted byinert groups. "Inert group" is understood here to mean a group whichdoes not react under the conditions encountered during the various stepsof the synthesis on a solid support of nucleic acids according to theinvention.

In a specific embodiment, a process for synthesizing polynucleotidescomprises the following steps of:

1) condensing the OH group in 5' or 3' of the first nucleotide or of anoligonucleotide linked at its other 3' or 5' end to the said solidsupport, by means of a coupling agent, with the phosphate groupoptionally substituted respectively in position 3' or 5' of a monomericnucleotide reagent protected in 3' and 5';

2) oxidizing or sulfurizing the phosphite type internucleotide linkageobtained in step 1) into a phosphate linkage respectively;

3) deprotecting the 5'--O or 3'--O end of the product obtained in step2);

4) repeating steps 1) to 3) as many times as there are nucleotides to beadded in order to synthesize the nucleic acid.

The above steps lead to an oligonucleotide linked to the solid support.The process comprises a final step of detaching the nucleic acid fromthe support and removing the groups for protecting the bases and, whereappropriate, the 2'--O positions of the nucleic acid.

In the techniques where the solid support is already linked to a firstnucleoside corresponding to the first nucleotide of the sequence to beprepared, before the start of the synthesis cycles, the said supportgenerally contains a protection in 5' or 3' of the said nucleoside. Inthis case, the synthesis cycle starts with a deprotection step in acidicmedium, in general a detritylation.

According to the variants used most commonly, the said monomericnucleotide reagent corresponds to the formula: ##STR1## in which: Arepresents H or an optionally protected hydroxyl group,

is a purine or pyrimidine base whose exocyclic amine functional group isoptionally protected,

C is a conventional protective group for the 5'--OH functional group,

x=0 or 1 with

a) when x=1:

R₃ represents H and R₄ represents a negatively charged oxygen atom, or

R₃ is an oxygen atom and R₄ represents either an oxygen atom or anoxygen atom carrying a protecting group, and

b) when x=0, R₃ is an oxygen atom carrying a protecting group and R₄ iseither a hydrogen or a disubstituted amine group,

when x is equal to 1, R₃ is an oxygen atom and R₄ is an oxygen atom, themethod is in this case the so-called phosphodiester method; when R₄ isan oxygen atom carrying a protecting group, the method is in this casethe so-called phosphotriester method,

when x is equal to 1, R₃ is a hydrogen atom and R₄ is a hydrogen atomand R₄ is a negatively charged oxygen atom, the method is in this casethe so-called H-phosphonate method, and

when x is equal to 0, R₃ is an oxygen atom carrying a protecting groupand R₄ is either a halogen, the method is in this case the so-calledphosphite method and, when R₄ is a leaving group of the disubstitutedamine type, the method is in this case the so-called phosphoramiditemethod.

The steps of a cycle of synthesis by the phosphoramidite method areconventionally the following:

1) condensation of the 5' terminal hydroxyl of a nucleoside or of anoligonucleotide covalently attached to the solid support with aphosphite type compound according to the reaction: ##STR2## 2) oxidationof the phosphite bond obtained to a phosphate according to the reaction:##STR3## 3) blocking of the hydroxyl groups of the unreactednucleosides; 4) liberation of the 5' terminal hydroxyl from the lastnucleoside so as to generate an active site for the next synthesiscycle.

Each nucleotide is sequentially added to the support by repeating steps1 to 4. At the end of the synthesis, the oligonucleotide is separatedfrom the support and freed of its protecting groups by a controlledhydrolysis reaction.

Commercial synthesizers specialized in the synthesis of oligonucleotidesare designed so as to automatically carry out the synthesis stepsdescribed above. These synthesizers are generally composed of a reactorcontaining the solid support, a reagent mixer, one or several unit(s)for selecting the reagents and the vessels containing the said reagents.The synthesis steps are carried out by successively adding the selectedreagents to the reactor. Most often, the solid support is washed withacetonitrile after each step.

The solid support is not immobilized and does not occupy the wholevolume of the reactor but, in general, only half of the volume of thereactor, and in any case no more than three quarters, so as to allowadequate stirring of the solid phase.

The reactor, as used in commercial synthesizers, has the shape of avessel crossed by the flow of reagents which causes the stirring (orfluidization) of the solid support. It is indeed considered that thestirring of the solid phase is essential because it allows betterpenetration of the solvents and reagents into the pores of the solidphase generally consisting of porous beads or porous membranes (seeMethods in Molecular Biology: Protocols for Oligonucleotides andAnalogs--Synthesis and Properties--Edited by SudhirAgrawal--1993--Humana Press, Totowa, N. J., pages 442-444 and 454).

The mixer is situated upstream of the reactor, connected to it by apipeline. The units for selecting the reagents generally consist ofelectrovalves and allow selective opening of the inlets/outlets of thehydraulic circuit as well as the routes for the passage of the reagents.The cohesion of the hydraulic system is ensured by a series of suitablyconnected tubes or capillaries. It is recommended to install theequipment in an airconditioned room at 20° C., the temperature at whichthe reactions are performed.

While the efficiency of the reactions is sufficient under theseconditions, the duration of the reactions and the required excess ofreagents in order to carry out good washing by successive dilutions arethe principal disadvantages of the synthesizers as described above. Inthis case, the consumptions of reagents and the duration of thesyntheses are substantially greater than the values calculated on thebasis of known laws of organic synthesis. These disadvantages wereattributed to the limiting factor which the rate of diffusion of thereagents in the pores constitutes.

It was discovered according to the present invention that it is mainlythe geometry of the vessel-type reactor in which the free particles ofthe solid support are stirred according to the principle of"pseudoliquid" phase reaction which has numerous disadvantages.

It was also discovered that several other parameters are also calledinto question which considerably reduce the productivity of thesynthesizers:

the reagents used for the oxidation step (iodine solution,tetrahydrofuran, lutidine, water) are degraded over time, and theresulting reduction in reactivity rapidly induces a reduction in theefficiency of the synthesis;

the absence of thermostatting of the reactor and of the reagents leadsto the loss of reproducibility of the syntheses.

The hydraulic system itself often comprises unusable dead volumes. Thetime spent by the reagents in these dead volumes and in the mixingchamber, in particular during the activation of phosphoramidite by thecoupling agent, limits the reactivity of the transient species formed.

Accordingly, the present invention relates to a set of modificationsmade to the system described above which has the effect of improving theproductivity by reducing the consumption of reagents and the durationsof synthesis.

The essential characteristic of the process according to the presentinvention relates to the immobilization of the solid phase or to themethod of filling the reactor with the solid phase.

The subject of the present invention is indeed a process for preparingpolynucleotides on a solid support in a reactor in the form of a columnthrough which solutions of reagents and/or solvents are circulated,wherein the solid phase constituting said solid support is immobilizedin said reactor, and said solutions migrate in the column and throughthe solid phase according to a frontal progression, such that thesuccessive solutions from each step of a synthesis cycle do not mix.

The subject of the present invention is also a process for preparingpolynucleotides on a solid support in a reactor in the form of a columnthrough which the solutions of reagents and/or solvents are circulated,wherein the reactor is a column completely filled with particles of aporous material constituting said solid support.

In this embodiment where the solid support consists of free particles ofa porous material filling a column, the particles are also immobilized.The immobilization indeed results from the fact that by "completelyfilled column" there is understood here that the degree of filling withparticles and the density of distribution of the particles in the columnare such that the particles are sufficiently packed such that they canno longer move when a liquid crosses the column.

It may be only a portion of the column which is completely filledprovided that the particles of the solid support are maintained byappropriate means in said portion of the column.

In an advantageous embodiment, the column will have a cylindrical shape.

It is thus proposed, for the first time, to use a "chromatographycolumn" type reactor, that is to say a column consisting of acylindrical tube completely filled with the solid phase.

This type of column, homogeneously filled with the solid support, offersseveral advantages: the various solutions which follow each other in thereactor do not mix, or very little, there is therefore no phenomenon ofdilution of the solutions by the previous reagent and the washingfollowing each step of the synthesis cycle is thereby greatlyfacilitated. According to the chromatography column principle, thesuccessive solutions introduced at one end of the column migrateaccording to a frontal progression, such that a solution "pushes" thepreceding one.

It was indeed observed that the solid phases conventionally used insolid phase oligonucleotide synthesis are such that the affinity and thephysicochemical interactions of the various reagents and solvents usedare negligable and do not interfere with their diffusion through thesolid phase other than by the desired chemical reactions.

It should be mentioned that, of course, in the interface region betweenthe solutions, slight mixing may occur locally between the solutionswhen the circulation rates of the solutions are high. However, thismixing, which is localized at the interface, is in no way comparablewith what is observed in prior processes for which the washes were basedon the principle of dilution by mixing successive solutions.

Contrary to the conventional model, these reactors therefore make itpossible to substantially reduce the volumes of reagents required. Theuseful flow rates are adjusted as a function of the diffusion constantsand the kinetics of the reactions called into play. The duration of thesynthesis cycles is thereby considerably reduced. For each type ofparticle, depending on its porosity, the flow rate of the reagentsthrough the column should not exceed a certain value above which thereagents no longer diffuse inside the pores. This is a well knownphenomenon in chromatography.

In addition, within the reactor and regardless of its size, theconditions are locally always identical. The system can therefore beapplied to all scales of synthesis.

In particular, there may be mentioned materials consisting of inorganicpolymers, especially glass, silica, metal oxides, or of organicpolymers, especially cellulose, or optionally substituted polystyrene.

Preferably, the polymer is an inorganic polymer prepared based on glassor silica, especially a silica gel.

The said particles may consist of microbeads or agglomeratedmicrofibers.

Preferably, in the reactor according to the present invention, there maybe used as particles of the solid support porous microbeads, therebyproviding the largest functionalized surface area in terms of weight ofnucleosides linked to the polymer per weight of polymers. The size ofthe beads may range from 5 μm to 500 μm, more generally from 40 μm to 70μm. The pores may optionally pass right through the microbeads. Thediameter of the pores may range between 200 Å and 10,000 Å. Two poresizes may be used, with wide pores (about 10,000 Å) and smaller pores(300 Å). This type of pore of variable size favors the diffusion of thesolutions through the solid phase.

The reactor according to the present invention makes it possible to bestexploit known laws of diffusion and kinetics of bimolecular chemicalreactions according to which the reagents in solution diffuse veryrapidly into the pores of the solid support and the kinetics ofcondensation of the free nucleoside with the 5' OH end of theoligonucleotide attached to the support is practically instantaneous.

The regulation of the temperature of the reactor and the reagents isanother essential characteristic of the present invention which, so far,does not exist in any synthesizer on the market.

The working temperature is one of the parameters which directly andsimultaneously influence the kinetics of the chemical reactions calledinto play, but also the stability of the reagents and the viscosity ofthe solutions. Contrary to all the prejudice which claims that thesynthesis of the oligonucleotides decreases the effeciency above 30° C.,it is recommended according to the present invention to thermostat thereactor at an optimal temperature of 45° C. The regulation of thetemperature of the system makes it possible to act on the kinetics ofthe chemical reactions called into play as well as on the viscosity ofthe solutions used.

Advantageously, the reagents, that is to say the coupling reagents suchas tetrazole, and the monomeric reagents, on the one hand, and/or thevarious reagents of the oxidizing solution, on the other hand, are mixedimmediately upstream of the reactor, or optionally, just upstream of asystem of distribution of the reagents in a multicolumn system. Theunusable dead volumes are thereby reduced.

In particular, the reagents are heated before introducing them into thereactor and, where appropriate, before mixing them.

Indeed, if the reagents are heated only after mixing them, a mutualinactivation and a loss of reactivity on the column are observed. Moreprecisely, early mixing (too far from the reactor) of the reagents canlead to undesirable side reactions. In particular, the mixing of thetetrazole (weak acid) and the phosphoramidites can result in thedetritylation of part of the monomers. The phosphoramidites can thenreact with each other. This type of reaction leads to the formation ofhomopolymers which may or may not react with the oligonucleotideattached to the support. The formation of oligonucleotides which arelonger than desired can be observed in this case. Whatever the case, thesynthesis yield is always thereby reduced. Of course, these phenomenaare accentuated by heating. It is therefore advisable to heat thereagents before mixing them.

In the reactors used before the present invention, above 30° C., thereagents become rapidly degraded. But, by means of the reactor accordingto the present invention, which favors the kinetics of the reactions, itis possible to increase the temperature up to 90° C. (between 20° and90° C.), preferably the temperature of the reactor is adjusted between30° and 60° C., especially to 45° C.

Advantageously, the deprotection step in acidic medium is performed withTFA (trifluoroacetic acid) in dichloromethane. A 1% v/v solution oftrifluoroacetic acid in extra dry dichloroethane is especially usedduring the step for liberation of the terminal 5' hydroxyl functionalgroup (step 4) by replacing the standard solution (2% dichloroaceticacid in dichloromethane). This recommendation makes it possible inparticular to get rid of the possible phenomena of boiling ofdichloromethane when the working temperature exceeds 40° C.

The mixing of acetic acid and pyridine causes an increase in thetemperatures from 40° to 60° C., which facilitates the maintenance ofthe temperature at said temperatures.

By virtue of the very high coupling yields (probably greater than 99%)of the process according to the invention, the step of blocking (or"capping") the unreacted 5'--OH functional groups (step 3) proves, inmost cases, not to be very efficient, or even completely pointless andcan therefore be omitted. Thus, no accumulation of synthetic fragmentsof size n-1 is observed as would be expected if no capping step isperformed. Probably, when the efficiency of the reactions is optimal,the unreacted species are permanently inaccessible.

According to another characteristic of the present invention, theoxidation step is performed with iodine in acetic acid and pyridine.Thus, in place of the conventional oxidation solution, two reagents areused separately, one consisting for example of an iodine solutionsaturated with glacial acetic acid, the other being ultrapure pyridine.These reagents are advantageous in that, at the oxidation step (step 2above), these two solutions can be mixed in stoichiometric quantities atthe inlet of the reactor. The mixture thus formed is injected into thereactor and instantaneously and quantitatively ensures the oxidation ofthe newly formed internucleotide bond. Thus, advantage is taken of thestability of the two separate solutions by indefinitely guaranteeing thereproducibility of the oxidation step.

The subject of the present invention is also a reactor for preparingpolynucleotides according to a process of the invention, the reactorbeing in the form of a column containing a solid support through whichthe solutions of reagents and/or solvents are circulated, wherein thesolid phase constituting the solid support is immobilized in saidreactor such that said solutions migrate in the column and through saidsolid phase according to a frontal progression, the successive solutionsof each step of a synthesis cycle not mixing at all or very little.

In particular, the subject of the present invention is therefore areactor which is useful in a process for preparing polynucleotides on asolid support, which consists of a cylindrical column completely filledwith particles of porous materials constituting a solid support asdescribed above.

The subject of the present invention is finally a device for thesynthesis of polynucleotides on a solid support containing athermostatted reactor and a thermostatted collector which performs thecollection, the heating to the temperature of the reactor, then themixing of the reagents before their introduction into the reactor.

Other advantages and characteristics of the present invention willappear in the light of the detailed description below.

Two oligonucleotide or polynucleotide synthesizers, designed on thebasis of the technical improvements described above, were produced. Theperformances and the productivity of these two apparatus are therebysubstantially increased compared to apparatus on the market.

The first synthesizer was designed to synthesize quantities ofoligonucleotides between 50 μmol and 1 mmol. These performances cantherefore be compared to those of commercial synthesizers which arepresent on the market and are capable of working at equivalent synthesisscales. This apparatus is called "LargeScale Synthesizer". It isrepresented in FIG. 1.

The function of the second apparatus is to synthesize, in parallel, 24different oligonucleotides or polynucleotides. The scale of eachsynthesis is between 0.05 and 1 μmol. So far, this apparatus has noequivalent. Its productivity can be compared to that of 6synthesizers--4 columns on the market. It is called "MulticolumnSynthesizer". It is represented in FIG. 2.

These two apparatus comprise:

the vessels (1) to (10)! containing the reagents, each comprising asyringe type positive displacement pumping module (11) to (20)!;

a thermostatted collector (21)! whose attributes are the collection, theheating and the regulation of the temperature and the mixing of themoving reagents.

The "LargeScale Synthesizer" is equipped with a "filled column" typereactor whose dimensions are adjusted as a function of the desiredsynthesis scale (22)!.

The "Multicolumn Synthesizer" is equipped with 24 microcolumns (22)! ofthe same type as the "LargeScale Synthesizer" but whose dimensions makeit possible to work at the scales 0.05 μmol, 0.1 μmol, 0.2 μmol and 1μmol. Between the collector and the reactors is a distributor (23)!responsible for equally distributing the reagents between the 24columns.

Finally, a block composed of 24 electrovalves, individually controlledat each column, makes it possible to select, at each synthesis step, thereactors "in service".

The volumes, the flow rates and the waiting times are under the controlof a computer. The program controls the correct sequence and theexecution of the cycles in conformity with the sequences to besynthesized.

In order to guarantee local conditions which are always identicalregardless of the number of reactors in service, the "MulticolumnSynthesizer" functions according to an interactive mode. Thus, thevolumes and the flow rates of each of the reagents are adjusted, at eachinstant, to the number of syntheses in progress.

A--Course of a synthesis with phosphoramidite in the LargeScaleSynthesizer

The synthesis scale is chosen at the time the program is initiated.

Each synthesis starts with an initialization cycle which makes itpossible to wash and thermostat the reactor to the working temperature.

The program comprises five synthesis cycles corresponding to each of thefour bases and to the possible modified bases.

The synthesis cycles can be broken down into synthesis steps orprocedures which follow one another in the following chronologicalorder:

1) Detritylation: liberation of the hydroxyl in position 5' of thenucleoside or of the oligonucleotide covalently attached to the solidsupport,

2) First washing;

3) Addition of the base: condensation of the phosphoramidite typemonomer with the free 5' terminal hydroxyl of the nucleoside or of theoligonucleotide attached to the solid support. Formation ofinternucleotide bond;

4) Second washing;

5) Oxidation of the previously formed phosphite type internucleotidebond into phosphate;

6) Third washing.

These cycles are repeated as many times as is required by theoligonucleotide synthesis or the synthesis of a polynucleotide ofdesired length.

B--Course of a synthesis in the Multicolumn Synthesizer

The synthesis scale is chosen at the time the program is initiated.

Each synthesis starts with an initialization cycle which makes itpossible to wash and thermostat the reactors to the working temperature.

There is only one synthesis cycle which is repeated as many times as isrequired by the manufacture of the 24 oligo- or polynucleotides.

The cycle can be broken down in the following manner:

a) selection of the columns to be detritylated.

1) Detritylation: liberation of the hydroxyl in position 5' of thenucleoside or of the oligonucleotide covalently attached to the solidsupport.

2) First washing:

b1) Selection of the columns which should receive the A base.

3-1) Addition of the A base: condensation of the phosphoramidite type Amonomer with the free 5' terminal hydroxyl nucleoside or of theoligonucleotide attached to the solid support. Formation of theinternucleotide bond.

b2) Selection of the columns which should receive the G base.

3-2) Addition of the G base: condensation of the phosphoramidite type Gmonomer with the free 5' terminal hydroxyl of the nucleoside or of theoligonucleotide attached to the solid support. Formation of theinternucleotide bond.

b3) Selection of the columns which should receive the T base.

3-3) Addition of the T base: condensation of the phosphoramidite type Tmonomer with the free 5' terminal hydroxyl of the nucleoside or of theoligonucleotide attached to the solid support. Formation of theinternucleotide bond.

b-4) Selection of the columns which should receive the C base.

3-4) Addition of the C base: condensation of the phosphoramidite type Cmonomer with the free 5' terminal hydroxyl of the nucleoside or of theoligonucleotide attached to the solid support. Formation of theinternucleotide bond.

c) Selection of the oxidizing columns.

4) Second washing.

5) Oxidation of the previously formed phosphite type internucleotidebond into phosphate.

6) Third washing.

DESCRIPTION OF THE STEPS MENTIONED ABOVE

Each step can be described with reference to the diagrams provided forthe synthesizers (FIGS. 1 and 2).

1) Detritylation: reagent no. 10, preferably a 1% v/v solution oftrifluoroacetic acid in extra dry dichloroethane, is pushed through thereactor by means of the syringe module no. 10.

The volume necessary is between 2 and 10 times the volume of the emptyreactor. The flow rate in the column is less than 500 cm/min. This flowrate unit in cm/min in fact represents the linear speed of the solutionsinside the column and is therefore independent of the diameter of thecolumn. The volume flow rates should be adjusted as a function of thediameter of the reactor. In order to change the size of the reactorwhile locally preserving the synthesis conditions, it is sufficient tomodify the volume flow rate while ensuring that the linear speed insidethe reactor, given in cm/min, does not change. The flow rates which areused are compatible with the diffusion constants in the pores, that isto say that the linear speed of the solutions inside a column is notgreater than the speed of diffusion through the pores. In the oppositecase, there would be a low efficiency of the reagents and of the washesresulting in a decrease in the synthesis yields. The total reaction timeis between 10 and 120 seconds.

2) First washing: the reagent no. 1, preferably extra dry acetonitrile,is pushed through the reactor by means of the syringe module no. 1. Thevolume required is between 2 and 10 times the volume of the emptyreactor. The flow rate in the column is less than 500cm/min.

3) Coupling: the reagents no. 3, or no. 4, or no. 5 or no. 6, or no. 7,and no. 2, respectively a 0.1 M solution of each monomer and a 0.45%solution of tetrazole in acetonitrile, are conjointly pushed through thereactor by means of the syringe modules no. 3, or no. 4, or no. 5, orno. 6, or no. 7, and no. 2 respectively. The ratios of the volumes andthe flow rates of the reagents no. 3, or no. 4, or no. 5, or no. 6, orno. 7, and no. 2 may be different from 1. The total volume required isbetween 2 and 10 times the volume of the empty reactor. The overall flowrate in the column is less than 500 cm/min. The reaction time is lessthan 1 minute.

4) Second washing: the reagent no. 1, preferably extra dry acetonitrile,is pushed through the reactor by means of the syringe module no. 1. Thevolume required is between 0.5 and 5 times the volume of the emptyreactor. The flow rate in the column is less than 500 cm/min.

5) Oxidation: the reagents no. 8 and no. 9, respectively a saturatedsolution of iodine in glacial acetic acid and ultrapure pyridine, areconjointly pushed through the reactor by means of the syringe modulesno. 8 and no. 9 respectively. The ratios of the volumes and flow ratesof the reagents no. 8 and no. 9 are equal to 1. The total volumerequired is between 1 and 5 times the volume of the empty reactor. Theoverall flow rate in the column is less than 500 cm/min. The reactiontime is less than 30 seconds.

6) Third washing: the reagent no. 1, preferably extra dry acetonitrile,is pushed through the reactor by means of the syringe module no. 1. Thevolume required is between 1 and 5 times the volume of the emptyreactor. The flow rate in the column is less than 500 cm/min.

The following exemplary embodiment sizes serve to illustrate the processaccording to the invention.

EXAMPLE 1: Synthesis of an oliconucleotide, at the 30 μmol scale, withthe aid of the LarqeScale Synthesizer

The apparatus used is that previously described.

The reactor used is a cylindrical glass column, of diameter 10 mm andheight 25 mm.

The working temperature is 45° C.

The synthesis cycles are detailed in Table 1.

An oligodeoxynucleotide, 18 bases long, whose sequence is: d(ACG TTC CTCCTG CGG GAA) is synthesized under these conditions.

The reactor is carefully filled with 0.66 g of CPG 500 Å (CPG INC.,USA), "derivatized" by the first A nucleoside. The capacity of thesupport is 45 μmol/g (density of 3 ml/g).

The synthesis scale is 30 μmol.

After a step of washing with acetonitrile, the synthesis cycle asdescribed in Table 1 is performed 17 times.

Under these conditions, the oligonucleotide, of the desired length,retains the transient dimethoxytrityl group at the 5' terminal position.

The oligonucleotide is separated from the CPG and liberated from thepermanent protecting groups by an appropriate treatment of the support,"derivatized" according to the method described above, with 10 ml of a30% aqueous solution of ammonium hydroxide, for 16 hours at 55° C.

After adding 40 ml of absolute ethanol and 1 ml of a 3M aqueous solutionof sodium acetate, the oligonucleotide is left to precipitate for twohours at 0° C.

The precipitate is then filtered on a 1.2 μlopridyne membrane (PALLS.A., FRANCE) and resolubilized in 10 ml of water.

After reading the optical density at 260 mm, 4000 O.D.U., that is to say120 mg of crude synthesis mixture, are obtained.

The purity of the oligonucleotide of the desired length, estimated byHPLC on a reversed-phase column, is 88%.

EXAMPLE 2: Synthesis of an oligonucleotide, at the 77 μmol scale, withthe aid of the LarqeScale Synthesizer

The apparatus used is the same as for the preceding example.

The reactor used is a cylindrical glass column, of diameter 10 mm andheight 25 mm.

The working temperature is 45° C.

The synthesis cycles are detailed in Table 2.

An oligodeoxynucleotide, 18 base in length, whose sequence is: d(TTC CGCCAG GAG GAA CGT) was synthesized under these conditions.

The reactor is carefully filled with 0.66 g of High-Loaded CPG 500 Å,"derivatized" by the first T nucleoside. The capacity of the support is110 μmol/g, its density 3 ml/g (MILLIPORE S.A., FRANCE).

The synthesis scale is 77 μmol.

After a step of washing with acetonitrile, we performed the synthesiscycle as described in Table 2 17 times.

Under these conditions, the oligonucleotide, of the desired length,retains the transient dimethoxytrityl group at the 5' terminal position.

The conditions for deprotection and recovery of the oligonucleotide arethe same as those described in the preceding example.

After reading the optical density at 260 nm, 8,500 O.D.U., that is tosay 255 mg of crude synthesis mixture, are obtained.

The purity of the oligonucleotide of the third length, estimated by HPLCon a reversed-phase column, is 84%.

Example 3: Synthesis of an oliconucleotide, at the 100 μmol scale, withthe aid of the LargeScale Synthesizer

The apparatus used is the same as for the previous example.

The reactor used is a cylindrical glass column, of diameter 15 mm andheight 34 mm.

The working temperature is 45° C.

The synthesis cycles are detailed in Table 3.

An oligonucleotide, 56 bases in length, whose sequence is d(TAA CCA CACTTT TTG TGT GGT TAA TGA TCT ACA GTT ATT TTT TAA CTG TAG ATC AT) issynthesized under these conditions.

The reactor is carefully filled with 2 g of CPG 500 Å, "derivatized" bythe first T nucleoside. The capacity of the support is 50 μmol/g, itsdensity 3 ml/g (MILLIPORE S.A., FRANCE).

The synthesis scale is 100 μmol.

After a step of washing with acetonitrile, we performed the synthesiscycle as is described in Table 3 55 times.

Under these conditions, the oligonucleotide, of the desired length,retains the transient dimethoxytrityl group at the 5' terminal position.

The conditions for deprotection and recovery of the oligonucleotide arethe same as those described in the previous examples.

After reading the optical density at 260 nm, 31,000 O.D.U., that is tosay 930 mg of crude synthesis mixture, are obtained.

The purity of the oligonucleotide of length 56 mers, estimated by HPLCon a reversed-phase column, is 61%.

Example 4: Simultaneous syntheses of 24 different oligonucleotides withthe aid of the Multicolumn Synthesizer

The reactors are metallic microcolumns of diameter 1.5 mm and height 6mm.

The working temperature is 50° C.

The synthesis cycles are detailed in Table 4.

We synthesized, under these conditons, the oligodeoxynucleotides whosesequences are given in Table 5.

The reactors are evenly filled with 2 mg of universal CPG 500 Åcontaining an epoxide group (prepared according to Example 1 of patentapplication FR 93 08 498). The capacity of the support is 50 μmol/g(GENSET S.A., FRANCE) (density: 3 ml/g).

The synthesis scale is 0.1 μmol.

After a step of washing with acetonitrile, we performed the operationsdescribed in Table 4 as many times as is required by the synthesis ofthe 24 oligodeoxynucleotides of Table 5.

The conditions for deprotection and recovery of the oligonucleotide arethe same as those described in the preceding examples.

After reading the optical density at 260 nm, 11 O.D.U. are obtained onaverage from each of the oligonucleotides, that is to say 0.33 mg. Thepurity of the oligonucleotides, estimated by HPLC, on a reversed-phasecolumn for the 5' O-Trityl oligodeoxynucleotides, and on an anionexchange column for the 5' OH oligonucleotides, exceeds 84%.

Comparative Example 5

Table 6 below presents the characteristics of the syntheses carried outwith fluidized bed reactors marketed by APPLIED BIOSYSTEMS and MILLIPOREas described in the literature.

The synthesizers used are the following:

APPLIED SYNTHESIZER, model 394:

reactor in the form of a column:

diameter 5 mm

height 6 mm

volume 0.11 ml

Solid support:

CPG 500 Å (CPG INC., USA)

capacity: 30 μmol/g

density: 3 ml/g

For a synthesis at the 0.2 μmol scale:

6.7 mg of CPG, that is to

say a volume of 0.02 ml and

a degree of filling of 20%.

MILLIPORE SYNTHESIZER, model 8800:

Reactor in the form of a 225 ml vessel which can be used with 1 to 15 gof CPG.

For our examples:

"STANDARD" method for 100 μmol

solid support:

CPG 500 Å (MILLIPORE S.A., FRANCE)

capacity: 30 μmol/g

density: 3 ml/g

For 100 μmol: 3.33 g of CPG that is to say 10 ml of phase and a degreeof filling of 4.5%.

"IMPROVED" method for 100 μmol

solid support:

HIGH-LOADED CPG 500 Å (MILLIPORE S.A., FRANCE)

capacity: 100 μmol/g

density: 3 ml/g

For 100 μmol:

1 g of CPG that is to say 3 ml of phase and a degree of filling of 1.3%.

In comparison with the results described in Examples 1 to 4, thedecrease in the duration of synthesis and the quantities of reagents isconsiderable. For the synthesis of 100 μmol:

with the process according to the invention (Example 3 and Table 3), 118ml of reagents and solvents are used and the duration of synthesis is 3minutes per synthesis cycle; with a MILLIPORE column, 500 ml of reagentsand solvents are used, and the duration of synthesis is 20 minutes percycle.

                                      TABLE 1    __________________________________________________________________________    SOLVENTS OR REAGENTS                  VESSEL NO                         VOLUME                               IND. FLOW RATE                                        TOTAL FLOW RATE                                                  TIME    __________________________________________________________________________    Dichloroethane, TFA 1% v/v                  10     20 ml 72 ml/min          17 sec    Waiting time                                  20 sec    Acetonitrile   1     8  ml 72 ml/min           7 sec    0.45M Tetrazole in acetonitrile                   2     3  ml 60 ml/min           3 sec    0.45M Tetrazole in acetonitrile                   2     6  ml 36 ml/min    plus                                48 ml/min 10 sec    0.1M monomer in acetonitrile                  3, 4, 5, 6 or 7                         2  ml 12 ml/min    Waiting time                                  20 sec    0.45M Tetrazole in acetonitrile                   2     1  ml 60 ml/min           1 sec    Acetonitrile   1     2  ml 60 ml/min           2 sec    Iodine sat. with Acetic Acid                   8     2.5                            ml 30 ml/min    plus                                60 ml/min  5 sec    Pyridine       9     2.5                            ml 30 ml/min    Acetonitrile   1     10 ml 60 ml/min          10 sec    TOTAL                57 ml                    1 min 35 sec    __________________________________________________________________________

                                      TABLE 2    __________________________________________________________________________                                       TOTAL FLOW    SOLVENTS OR REAGANTS                  VESSEL NO.                         VOLUME                              IND. FLOW RATE                                       RATE   TIME    __________________________________________________________________________    Dichloroethane, TFA 1% v/v                  10     20 ml                              72 ml/min       17 sec    Waiting time                              30 sec    Acetonitrile   1     8  ml                              72 ml/min        7 sec    0.45M Tetrazole in acetonitrile                   2     3  ml                              60 ml/min        3 sec    0.45M Tetrazole in acetonitrile                   2     6  ml                              36 ml/min    plus                               48.6 ml/min                                              10 sec    0.1M monomer in acetonitrile                  3, 4, 5, 6 or 7                         2.1                            ml                              12.6 ml/min    Waiting time                              40 sec    0.45M Tetrazole in acetonitrile                   2     1  ml                              60 ml/min        1 sec    Acetonitrile   1     2  ml                              60 ml/min        2 sec    Iodine sat. with Acetic Acid                   8     2.5                            ml                              30 ml/min    plus                               60 ml/min                                               5 sec    Pyridine       9     2.5                            ml                              30 ml/min    Acetonitrile   1     10 ml                              60 ml/min       10 sec    TOTAL                57.1                            ml                2 min 05 sec    __________________________________________________________________________

                                      TABLE 3    __________________________________________________________________________    SOLVENTS OR REAGENTS                  VESSEL NO                         VOLUME                               IND. FLOW RATE                                        TOTAL FLOW RATE                                                  TIME    __________________________________________________________________________    Dichloroethane, TFA 1% v/v                  10     40 ml 84 ml/min          30 sec    Waiting time                                  30 sec    Acetonitrile   1     14 ml 72 ml/min          15 sec    0.45M Tetrazole in acetonitrile                   2     6  ml 60 ml/min           6 sec    0.45M Tetrazole in acetonitrile                   2     14 ml 36 ml/min    plus                                48 ml/min 25 sec    0.1M monomer in acetonitrile                  3, 4, 5, 6 or 7                         5  ml 12 ml/min    Waiting time                                  45 sec    0.45M Tetrazole in acetonitrile                   2     3  ml 60 ml/min           3 sec    Acetonitrile   1     6  ml 60 ml/min           6 sec    Iodine sat. with Acetic Acid                   8     5  ml 30 ml/min    plus                                60 ml/min 10 sec    Pyridine       9     5  ml 30 ml/min    Acetonitrile   1     20 ml 60 ml/min          15 sec    TOTAL                118                            ml                    3 min 05 sec    __________________________________________________________________________

                                      TABLE 4    __________________________________________________________________________                      Dichloro-    0.45M        Sol. of                      ethane                           Ace-    Tetrazole Ace-                                                Iodine    TOTAL                                                               TIME/                      1% v/v                           toni-                              0.45M                                   0.1M 0.45M                                             toni-                                                sat.      VOLUME                                                               CYCLE                      TFA  trile                              Tetrazole                                   Monomers                                        Tetrazole                                             trile                                                Pyridine                                                    Acetonitrile                                                          (ml) (min)    __________________________________________________________________________    STEP              Detrityl-                           Wash                              Coupling                                   Coupling                                        Wash 2                                             Wash                                                Oxida-                                                    Wash 3                      ation                           1                 2  tion    VESSEL NO.        10   1  2    3, 4, 5, 6                                        2    1  8 and 9                                                    1                                   or 7 and 2     1 COLUMN           VOLUME (ml)                      0.8  0.4                              0.0375                                   0.08 0.025                                             0.4                                                0.1 0.4   2.2425           FLOW RATE (ml/min)                      6    4.8                              2.25 2.94 1.5  4.8                                                3   4.8        0.48     2 COLUMNS           VOLUME (ml)                      1.2  0.6                              0.075                                   0.16 0.05 0.6                                                0.15                                                    0.6   3.425           FLOW RATE (ml/min)                      9    7.2                              4.5  2.94 3    7.2                                                3.15                                                    7.2        0.52     3 COLUMNS           VOLUME (ml)                      1.6  0.8                              0.1125                                   0.24 0.075                                             0.8                                                0.2 0.8   4.6275           FLOW RATE (ml/min)                      12   9.6                              6.75 5.88 4.5  9.6                                                3.3 9.6        0.52     4 COLUMNS           VOLUME (ml)                      2    1  0.15 0.32 0.1  1  0.25                                                    1     5.82           FLOW RATE (ml/min)                      15   12 9    8.82 6    12 3.45                                                    12         0.53     5 COLUMNS           VOLUME (ml)                      2.4  1.2                              0.1875                                   0.4  0.125                                             1.2                                                0.3 1.2   7.0125           FLOW RATE (ml/min)                      18   14.4                              11.25                                   11.76                                        7.5  14.4                                                3.6 14.4       0.53     6 COLUMNS           VOLUME (ml)                      2.8  1.4                              0.225                                   0.48 0.15 1.4                                                0.35                                                    1.4   8.205           FLOW RATE (ml/min)                      21   16.8                              13.5 14.7 9    16.8                                                3.75                                                    16.8       0.54     7 COLUMNS           VOLUME (ml)                      3.2  1.6                              0.2625                                   0.56 0.175                                             1.6                                                0.4 1.6   9.3975           FLOW RATE (ml/min)                      24   19.2                              15.75                                   17.64                                        10.5 19.2                                                3.9 19.2       0.55     8 COLUMNS           VOLUME (ml)                      3.6  1.8                              0.3  0.64 0.2  1.8                                                0.45                                                    1.8   10.59           FLOW RATE (ml/min)                      27   21.6                              18   20.58                                        12   21.6                                                4.05                                                    21.6       0.56     9 COLUMNS           VOLUME (ml)                      4    2  0.3375                                   0.72 0.225                                             2  0.5 2     11.7825           FLOW RATE (ml/min)                      30   24 20.25                                   23.52                                        13.5 24 4.2 24         0.57    10 COLUMNS           VOLUME (ml)                      4.4  2.2                              0.375                                   0.8  0.25 2.2                                                0.55                                                    2.2   12.975           FLOW RATE (ml/min)                      33   26.4                              22.5 26.46                                        15   26.4                                                4.35                                                    26.4       0.57    11 COLUMNS           VOLUME (ml)                      4.8  2.4                              0.4125                                   0.88 0.275                                             2.4                                                0.6 2.4   14.1675           FLOW RATE (ml/min)                      36   28.8                              24.75                                   29.4 16.5 28.8                                                4.5 28.8       0.58    12 COLUMNS           VOLUME (ml)                      5.2  2.6                              0.45 0.96 0.3  2.6                                                0.65                                                    2.6   15.36           FLOW RATE (ml/min)                      39   31.2                              27   32.34                                        18   31.2                                                4.65                                                    31.2       0.59    13 COLUMNS           VOLUME (ml)                      5.6  2.8                              0.4875                                   1.04 0.325                                             2.8                                                0.7 2.8   16.5525           FLOW RATE (ml/min)                      42   33.6                              29.25                                   35.28                                        19.5 33.6                                                4.8 33.6       0.59    14 COLUMNS           VOLUME (ml)                      6    3  0.525                                   1.12 0.35 3  0.75                                                    3     17.745           FLOW RATE (ml/min)                      45   36 31.5 38.22                                        21   36 4.95                                                    36         0.60    15 COLUMNS           VOLUME (ml)                      6.4  3.2                              0.5625                                   1.2  0.375                                             3.2                                                0.8 3.2   18.9375           FLOW RATE (ml/min)                      48   38.4                              33.75                                   41.16                                        22.5 38.4                                                5.1 38.4       0.60    16 COLUMNS           VOLUME (ml)                      6.8  3.4                              0.6  1.28 0.4  3.4                                                0.85                                                    3.4   20.13           FLOW RATE (ml/min)                      51   40.8                              36   44.1 24   40.8                                                5.25                                                    40.8       0.61    17 COLUMNS           VOLUME (ml)                      7.2  3.6                              0.6375                                   1.36 0.425                                             3.6                                                0.9 3.6   21.3225           FLOW RATE (ml/min)                      54   43.2                              38.25                                   47.04                                        25.5 43.2                                                5.4 43.2       0.61    18 COLUMNS           VOLUME (ml)                      7.6  3.8                              0.675                                   1.44 0.45 3.8                                                0.95                                                    3.8   22.515           FLOW RATE (ml/min)                      57   45.6                              40.5 49.98                                        27   45.6                                                5.55                                                    45.6       0.62    19 COLUMNS           VOLUME (ml)                      8    4  0.7125                                   1.52 0.475                                             4  1   4     23.7075           FLOW RATE (ml/min)                      60   48 42.75                                   52.92                                        28.5 48 5.7 48         0.62    20 COLUMNS           VOLUME (ml)                      8.4  4.2                              0.75 1.6  0.5  4.2                                                1.05                                                    4.2   24.9           FLOW RATE (ml/min)                      63   50.4                              45   55.86                                        30   50.4                                                5.85                                                    50.4       0.62    21 COLUMNS           VOLUME (ml)                      8.8  4.4                              0.7875                                   1.68 0.525                                             4.4                                                1.1 4.4   26.0925           FLOW RATE (ml/min)                      66   52.8                              47.25                                   58.8 31.5 52.8                                                6   52.8       0.63    22 COLUMNS           VOLUME (ml)                      9.2  4.6                              0.825                                   1.76 0.55 4.6                                                1.15                                                    4.6   27.285           FLOW RATE (ml/min)                      69   55.2                              49.5 61.74                                        33   55.2                                                6.15                                                    55.2       0.63    23 COLUMNS           VOLUME (ml)                      9.6  4.8                              0.8625                                   1.84 0.575                                             4.8                                                1.2 4.8   28.4775           FLOW RATE (ml/min)                      72   57.6                              51.75                                   64.68                                        34.5 57.6                                                6.3 57.6       0.64    24 COLUMNS           VOLUME (ml)                      10   5  0.9  1.92 0.6  5  1.25                                                    5     29.67           FLOW RATE (ml/min)                      75   60 54   67.62                                        36   60 6.45                                                    60         0.64    __________________________________________________________________________

                  TABLE 5    ______________________________________    COLUMN          5'     No    NO.    OLIGO    Trityl bases                                5'-3' SEQUENCE    ______________________________________     0     OLIGO1   YES    12   TTT TTT TTT TTT     1     OLIGO2   NO     12   TTT TTT TTT TTT     2     OLIGO3   YES    15   TTT TTT TTT TTT TTT     3     OLIGO4   NO     15   TTT TTT TTT TTT TTT     4     OLIGO5   YES    18   TTT TTT TTT TTT TTT TTT     5     OLIGO6   NO     18   TTT TTT TTT TTT TTT TTT     6     OLIGO7   YES    12   AAA AAA AAA AAA     7     OLIGO8   NO     12   AAA AAA AAA AAA     8     OLIGO9   YES    15   AAA AAA AAA AAA AAA     9     OLIGO10  NO     15   AAA AAA AAA AAA AAA    10     OLIGO11  YES    18   AAA AAA AAA AAA AAA AAA    11     OLIGO12  NO     18   AAA AAA AAA AAA AAA AAA    12     OLIGO13  YES    12   CCC CCC CCC CCC    13     OLIGO14  NO     12   CCC CCC CCC CCC    14     OLIGO15  YES    15   CCC CCC CCC CCC CCC    15     OLIGO16  NO     15   CCC CCC CCC CCC CCC    16     OLIGO17  YES    18   CCC CCC CCC CCC CCC CCC    17     OLIGO18  NO     18   CCC CCC CCC CCC CCC CCC    18     OLIGO19  YES    12   AGT CAG TCA GTC    19     OLIGO20  NO     12   AGT CAG TCA GTC    20     OLIGO21  YES    15   AGT CAG TCA GTC AGT    21     OLIGO22  NO     15   AGT CAG TCA GTC AGT    22     OL1GO23  YES    18   AGT CAG TCA GTC AGT CAG    23     OL1GO24  NO     18   AGT CAG TCA GTC AGT CAG    ______________________________________

                  TABLE 6    ______________________________________                              MILLI-                              PORE     MILLIPORE                              LARGE-   LARGE-    SYNTHESIZERS   ABI 394    SCALE    SCALE    CHARACTERISTICS                   4 COLUMNS  100 μmol                                       100 μmol    SYNTHESIS SCALE                   0.2 μmol                              STAND-   IM-    METHOD         STANDARD*  ARD**    PROVED**    ______________________________________    REAGENTS       (ml)       (ml)     (ml)    MONOMERS 0.1M  0.110      5        2.62    TETRAZOLE SOLUTION                   0.400      20.3     19    CAP A (acetic anhydride)                   0.290      9        6.25    CAP B (N-methylimidazole)                   0.260      11.6     8.65    DEBLOCK (TCA/DCM)                   1.100      84.3     68.75    IODINE SOLUTION                   0.350      40       33    ACETONITRILE   8.000      376      286.25    TOTAL          10,510     546.2    424.52    TIME PER CYCLE 6 min      20 min   22 min    ______________________________________     *APPLIED BIOSYSTEMS Instruction manual for the ABI 394 synthesizer     **N.D. SINHA, S.FRY; 3rd CAMBRIDGE SYMPOSIUM Oligonucleotides & Analogues     5-8 September 1993 POSTER COMMUNICATION

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 31    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..18    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ACGTTCCTCCTGCGGGAA18    ThrPheLeuLeuArgGlu    15    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    ThrPheLeuLeuArgGlu    15    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..18    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TTCCGCCAGGAGGAACGT18    PheArgGlnGluGluArg    15    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    PheArgGlnGluGluArg    15    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 56 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..56    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    TAACCACACTTTTTGTGTGGTTAATGATCTACAGTTATTTTTTAACTG48    *ProHisPheLeuCysGly**SerThrValIlePhe*Leu    151015    TAGATCAT56    *Ile    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    ProHisPheLeuCysGly    15    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    SerThrValIlePhe    15    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..12    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    TTTTTTTTTTTT12    PhePhePhePhe    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    PhePhePhePhe    1    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..15    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    TTTTTTTTTTTTTTT15    PhePhePhePhePhe    15    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    PhePhePhePhePhe    15    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..18    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    TTTTTTTTTTTTTTTTTT18    PhePhePhePhePhePhe    15    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    PhePhePhePhePhePhe    15    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..12    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    AAAAAAAAAAAA12    LysLysLysLys    1    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    LysLysLysLys    1    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..15    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    AAAAAAAAAAAAAAA15    LysLysLysLysLys    15    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    LysLysLysLysLys    15    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..18    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    AAAAAAAAAAAAAAAAAA18    LysLysLysLysLysLys    15    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    LysLysLysLysLysLys    15    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..12    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    CCCCCCCCCCCC12    ProProProPro    1    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    ProProProPro    1    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..15    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    CCCCCCCCCCCCCCC15    ProProProProPro    15    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    ProProProProPro    15    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..18    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    CCCCCCCCCCCCCCCCCC18    ProProProProProPro    15    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    ProProProProProPro    15    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..12    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    AGTCAGTCAGTC12    SerGlnSerVal    1    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    SerGlnSerVal    1    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..15    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    AGTCAGTCAGTCAGT15    SerGlnSerValSer    15    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    SerGlnSerValSer    15    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..18    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    AGTCAGTCAGTCAGTCAG18    SerGlnSerValSerGln    15    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    SerGlnSerValSerGln    15    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We claim:
 1. A process for preparing polynucleotides on a solid supportin a reactor, in the form of a column containing said solid support,through which solutions of reagents and/or solvents are circulated,wherein a solid phase constituting said solid support is immobilized insaid reactor, and wherein said solutions migrate in the column andthroughout the solid phase according to a frontal progression, such thatsuccessive solutions from each step of a synthesis cycle do not mix, andwherein the reactor is maintained at a temperature between 30° C. and90° C.
 2. The process as in claim 1, wherein the reactor is maintainedat a temperature between 30° C. and 60° C.
 3. The process as is in claim1, wherein the reactor is maintained at a temperature on the order of45° C.
 4. The process of claim 1, wherein the column is cylindrical, andwherein the solid support comprises particles of a porous material. 5.The process of claim 1, wherein the solid support comprises particles ofa porous material, which particles are motionless during the process. 6.The process of claim 4, wherein the particles of a porous material aremotionless during the process.
 7. The process as in claim 1, wherein thesolid phase is composed of porous microbeads.
 8. The process as in claim1, wherein the reagents are heated before introducing them into saidreactor.
 9. The process of claim 8, wherein the reagents are heatedbefore mixing them.
 10. The process as in claim 8, wherein the reagentsare heated to the temperature of the reactor.
 11. The process as inclaim 9, wherein the reagents are heated to the temperature of thereactor.
 12. The process as in claim 1, wherein a deprotection step iscarried out with TFA in dichloroethane.
 13. The process as in claim 1,wherein an oxidation step is carried out with iodine in acetic acid andpyridine.